Project Summary: The peptidoglycan cell wall of gram-positive bacteria remains an active element of the bacterial cell, utilizing a conserved and defined set of late cell wall biosynthesis proteins that are required for the cross-linking of specific stem peptide species, creating a delicate array of specific cross-links. These cross-links allow the cell wall to act as a mesh encasement, making the cell tolerant to osmotic lysis, and therefore permitting cell survival. Of particular interest are novel and essential proteins required for murein biosynthesis in gram- positive pathogenic bacteria, such as Streptococcus pneumoniae, as new antimicrobial targets are needed in order to produce more effective antibiotics. One such protein is the partially characterized and essential putative cell wall transpeptidase, PcsB. The focus of this work is to determine the function of PcsB in S. pneumoniae, which will be accomplished with two specific aims. The first aim is to localize PcsB during the cell cycle. This will be accomplished with immunofluorescence methods, utilizing single and co-localization tactics, as well as immunoblot techniques, and will serve to set a time of action for PcsB as well as suggest possible interacting proteins. The second aim is to biochemically define the function of PcsB by examining the extent of cross-linking in an in vitro atmosphere by means of reverse-phase high pressure liquid chromatography and mass spectrometry methods. Understanding both the localization patterns of PcsB in pneumococcus during the cell cycle, as well as determining the biochemical function of this essential protein will help to define our knowledge of cell wall biosynthesis in the gram-positive human pathogen, S. pneumoniae. Relevance: S. pneumoniae is a gram-positive bacterial pathogen mainly of the human respiratory tract, infection of which can result in several diseases, including sinusitis, meningitis, pneumonia, septicemia, and otitis media, making S. pneumoniae an important bacteria for biomedical research. In the past four decades, pneumococcal diseases have been on the rise with resistance to [unreadable]-lactam antibiotics and other commonly used anti-microbial drugs, therefore creating an ever-present need to find new ways to combat this human pathogen. One way to determine new protein targets for antimicrobial drugs is the study of cell wall biosynthesis in S. pneumoniae, particularly the study of proteins essential to the viability of the organism, such as PcsB.